Following initiation of the clotting process, blood coagulation proceeds through the sequential activation of certain plasma proenzymes to their enzyme forms. These plasma glycoproteins, including Factor XII, Factor XI, Factor IX, Factor X, Factor VII, and prothrombin, are zymogens of serine proteases. Most of these blood clotting enzymes are effective on a physiological scale only when assembled in complexes on membrane surfaces with protein cofactors such as Factor VIII and Factor V. Other blood factors modulate and localize clot formation, or dissolve blood clots. Activated protein C is a specific enzyme that inactivates procoagulant components. Calcium ions are involved in many of the component reactions. Blood coagulation follows either the intrinsic pathway, where all of the protein components are present in blood, or the extrinsic pathway, where the cell-membrane protein tissue factor plays a critical role. Clot formation occurs when fibrinogen is cleaved by thrombin to form fibrin. Blood clots are composed of activated platelets and fibrin.
Thrombin is a multifunctional protease that regulates several key biological processes. For example thrombin is among the most potent of the known platelet activators. In addition, as noted above, thrombin is essential for the cleavage of fibrinogen to fibrin to initiate clot formation. These two elements are involved in normal hemostasis but in atherosclerotic arteries can initiate the formation of a thrombus, which is a major factor in pathogenesis of vasoocclusive conditions such as myocardial infarction, unstable angina, nonhemorrhagic stroke and reocclusion of coronary arteries after angioplasty or thrombolytic therapy. Thrombin is also a potent inducer of smooth cell proliferation and may therefore be involved in a variety of proliferative responses such as restenosis after angioplasty and graft induced atherosclerosis. In addition, thrombin is chemotactic for leukocytes and may therefore play a role in inflammation, Hoover, R. J. et al., Cell 14:423 (1978); Etlngin, O. R. et al., Cell 61:657 (1990). These observations indicate that inhibition of thrombin formation or inhibition of thrombin itself may be effective in preventing or treating thrombosis, limiting restenosis and controlling inflammation.
The formation of thrombin is the result of the proteolytic cleavage of its precursor prothrombin at the Arg-Thr linkage at positions 271-272 and the Arg-Ile linkage at positions 320-321. This activation is catalyzed by the prothrombinase complex, which is assembled on the membrane surfaces of platelets, monocytes, and endothelial cells. The complex consists of Factor Xa (a serine protease), Factor Va (a cofactor), calcium ions and the acidic phospholipid surface. Factor Xa is the activated form of its precursor, Factor X, which is secreted by the liver as a 58 kDa precursor and is converted to the active form, Factor Xa, in both the extrinsic and intrinsic blood coagulation pathways. It is known that the circulating levels of Factor X, and of the precursor of Factor Va, Factor V, are on the order of 10-7M. Them has been no determination of the levels of the corresponding active Factors Va and Xa.
The amino acid sequences and genes of most of the plasma proteins involved in hemostasis of blood are commonly known, such as Factor IIa, Factor Va, Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, Activated Protein C, Activated Protein S, fibrinogen and thrombin. Also commonly known am the amino acid sequences and genes of the precursor forms of these blood factors, and common methods for their activation or conversion to mature forms.
Factor X (Stuart Factor) is an essential component of the blood coagulation cascade (see, FIGS. 1 and 2). Factor X is a member of the calcium ion binding, gamma carboxyglutamyl ("Gla")-containing, vitamin K dependent, blood coagulation glycoprotein family, which also includes Factors VII and IX, prothrombin, protein C and protein S, Furie, B. et al., Cell 53:505 (1988). Factor X is the zymogen for the serine protease Factor Xa. Factor Xa combines with a co-factor, activated Factor V, calcium, and phospholipids on a membrane surface to form the prothrombinase complex. This enzyme complex converts prothrombin to thrombin, which then converts fibrinogen to fibrin, one of the pathways resulting in thrombosis (Colman, R. W. et al., "Overview of Hemostasis" in Colman, R. W., et al., Hemostatis and Thrombosis, Basic Principles and Clinical Practice, Second Edition (1987), Part I, Section A, Plasma Coagulation Factors, pp. 3-17).
Factor X can be purified from natural, synthetic or recombinant sources by any of a number of different extractive and chromatographic techniques, such as: a combination of ion-exchange, heparin-affinity and hydroxylapatite chromatography (Kosow, D. P., Thromb. Res., 9(6):565-573 (1976); sulfated dextran (Miletich, J. P. et al., Analytical Biochemistry 105:304-310 (1980)); a combination of barium citrate adsorption, ammonium sulfate precipitation, ion-exchange and heparin-affinity chromatography (Bajaj, S. P. et al., Prep. Biochem 11:397-412 (1981)); Cohn fractionation (Monohan, J. B. et al., Thromb. Res. 19(6):743-755 (1980)); sulfated non-carbohydrate matrices (U.S. Pat. Nos. 4,721,572; and 4,725,673); immunoaffinity chromatography (European Patent Application 0 286,323); hydrophobic interaction chromatography (Freidberg, R. C., et al., Prep. Biochem. 18(3):303-320 (1988) ); metal-chelate chromatography (PCT/GB88/01150); a combination of immunoaffinity and ion-exchange (Ahmad, S. S. et al., Thromb. Res. 55(1):121-133 (1989)); and as a by-product in the purification of other blood coagulation factors (Hrinda, M. E., et al., "Preclinical Studies of a Monoclonal Antibody-Purified Factor IX; Mononine.TM.," in Seminars in Hematology 28(3) Suppl. 6:6-14 (1991); and U.S. Pat. No. 5,071,961.) Typically, Factor X activation, inactivation and purification are accomplished separately.
Factor X must be activated to Factor Xa before the protease is incorporated into the prothrombinase complex (Steinberg, M. et al., "Activation of Factor X" in Colman, R. W. et al., supra, Part I, Section A, Chapter 7, pp 112-119). Factor Xa is a two chain molecule linked by one disulfide bond between the two chains. The heavy chain contains the serine protease, trypsin-like active site and the N-terminal activation peptide which is glycosylated. The heavy chain has at least three forms, a, .beta. and g, which differ due to the cleavage of a C-terminal peptide in the heavy chain (Aronson, D. L. et al., Proc. Soc. Exp. Biol. Med. 137(4):1262-1266 (1971); Mertens, K. et al., Biochem J. 185:647-658 (1980)). This C-terminal peptide is thought to be glycosylated through an O-linked type glycosylation. The a form is the full length form of the heavy chain and the .beta. and g forms are clipped. The light chain contains a growth factor-like domain and a number of unique post-translationally modified amino acid residues, called gamma-carboxy glutamic acid residues ("GLA's") which are implicated in imparting activity through calcium binding interactions required in the prothrombinase complex (Davie, E. W., "The Blood Coagulation Factors: Their cDNAS, Genes and Expression" in Colman, R. W. et al., supra, Part I, Section A, pp. 242-268).
Factor X can be activated to Factor Xa by any of several methods. Factor X is activated naturally through the extrinsic pathway (Factor VIIa/Tissue Factor complex) or the intrinsic pathway (Factor VIIIa/Factor Ixa-phospholipid-calcium enzyme complex) (Mertens, K. et al., Biochem J. 185:647-658 (1980); Jesty, J., J. Biol. Chem. 261(19):8695-8702 (1986); Steinberg, M. et al., supra; Bauer, K. et al., Blood 74(6):2007-2015 (1989); Chattopadhyay, A. et al., J. Biol. Chem. 2:735-739 (1989)). Factor X can also be activated to Factor Xa by proteases such as Russell's Viper Venom Factor X activating enzyme ("RVV-X") (Furie, B. C. et al., Methods in Enzymology 45:191-205 (1976); DiScipio, R. G. et al., Biochemistry 16(24):5253-5260 (1977); trypsin (Steinberg, M., et al., supra); or cancer procoagulant (Gordon, S. G. et al., Blood Coagulation and Fibrinolysis 2:735-739 (1991)).
It is known that numerous snake venom activities affect the intrinsic coagulation mechanism by variously activating, inhibiting or converting factors in the blood coagulation cascade; snake venoms are known which activate Protein C, prothrombin, thrombin-like enzymes, fibrinogenases, and activities of Factors V and X (N. A. Marsh, Blood Coagulation and Fibrinolysis 5:399-410 (1994). Synthetic peptides and peptidomimetics are also known as substrates and inhibitors of serine proteases (Claeson, G., Blood Coagulation and Fibrinolysis 5:411-436 (1994). A number of general and specific serine protease inhibitors are also known.
Various activators and inhibitors are commonly known for many of the blood factors. For example, Factor I (fibrinogen) is known to be activated by thrombin; Factor II (prothrombin) is known to be activated by Factor Xa and thrombin; Factor V is known to be activated by papain, a Factor-V-activation protease from Russell viper venom, plasmin, Factor Xa, chymotrypsin, and thrombocytin, and is inactivated by activated Protein C; Factor VII is known to be activated by minor proteolysis, with a signal peptidase and a processing protease; Factor IX is known to be activated by Factor XIa with calcium ions, tissue factor, Factor VII, and Russell viper venom-X, and is known to be inactivated by hirudin and antithrombin III; Factor X is activated by Factors IXa and VII with phospholipid and calcium ions, and by Russell viper venom; Factor XI is known to be activated by Factor XIIa and trypsin; Factor XII is known to be activated by contact with negatively charged surfaces, sulfatides, trypsin, plasmin, and kallikrein; Protein C is activated by thrombin, etc. See, Colman et al., supra. for the text describing known blood factor activators and inactivators.
In some circumstances, it is desirable to interfere with the functioning of Factor Xa in order to prevent excessive clotting. In other circumstances, such as in hemophilia, it is desirable to provide a source of Factor Xa independent of the activation process that takes place in normal individuals. Both of the common forms of hemophilia (hemophilia A and B) involve deficiencies in only the intrinsic pathway of activation, but the operation of the extrinsic pathway does not appear to be successful in arresting bleeding. Similarly, other patients are treated currently for deficiencies of other blood factors (such as VII, X, XI, XIII), or yon Willebrand's disease. Factor VII deficiency is not as clinically well-defined as hemophilia A or B, however patients with Factor VII deficiency have been reported to have extensive bleeding. Protein C deficiency is associated with thrombotic risk.
Factor Xa, and several other activated blood factors, have typically not been useful as pharmaceuticals because of their extremely short half-life in serum, which for example typically is only about 30 seconds for Factor Xa. Use of acylation to prolong the half-life of certain blood factors has been disclosed. For example, Casseis, R. et al., Biochem. Jour. 247:359-400 (1987), reports that various acylating agents remained bound to urokinase, tPA and streptokinase-plasminogen activator complex for time periods ranging from a half-life of 40 minutes to a half-life of over 1,000 minutes depending on the nature of the acylating group and the nature of the factor. U.S. Pat. No. 4,337,244 describes acylation of tPA or streptokinase. Use of an amidinophenyl group functioning as an arginine analog to introduce, temporarily, a substituted benzoyl group into the active site for the purpose of enhancing serum stability was discussed by Fears, R. et al., Seminars in Thrombosis and Homeostasis 15:129-39 (1980) (see also: Fears, R. et al., Drugs 33 Suppl. 3:57-63 (1987); Sturzebecher, J. et al., Thrombosis Res. 47:699-703 (1987)), which describes stabilized acyl derivatives of tPA. Use of the acylated plasminogen streptokinase activator complex ("APSAC") is described in Crabbe, S. J. et al., Pharmacotherapy 10:115-26 (1990). Acylated forms of thrombin have also been described. Generally, methods for activating, inhibiting, and recovering the target blood factor have been multi-step and complex processes.
Chemically inactivated forms of Factor Xa can be used in a number of therapeutic indications (U.S. Pat. Nos. 4,285,932; 5,120,537; Benedict, C. R. et al., Blood 81(8):2059-2066 (1993); U.S. Ser. No. 08/268,003, filed Jun. 26, 1994; Sinha, U. et al., "Procoagulation Activities of Reversibly Acylated forms of Factor Xa," presented at the 35th Annual Meeting of the American Heart Association, St. Louis, Mo., Dec. 3-7, 1993). Factor Xa can be irreversibly inactivated using chloromethyl ketone derivatives, such as glutamyl glycyl arginyl ("EGR") chloromethyl ketone, or dansyl glutamyl glycyl arginyl ("DEGR") chloromethyl ketone (see e.g.: Nesheim, H. E. et al., Jour. Biol. Chem. 254:10952 (1979); U.S. Pat. No. 5,120,537; Kettner, C. et al., Biochem 17(22):4778-4783 (1978); Kettner, C. et al., Biochim. Biophys. Acta. 569:31-40 (1979); Kettner, C. et al., Arch. Biochem. Biophys. 202:420-430 (1980); Kettner, C. et al., Methods in Enzymology 80 Part C:826-842 (1981); Kettner, C. et al., Thromb. Res. 22:645-652 (1981); Nesheim, M. E. et al., J. Biol. Chem. 256(13):6537-6540 (1981); U.S. Pat. No. 4,318,904; Lijnen, H. R. et al., Thromb. Res. 34:431-437 (1984); Williams, B., et al., J. Biol. Chem. 264(13):7536-7545 (1989); U.S. Pat. No. 5,153,175). This irreversibly inactivated Factor Xa can be used to inhibit thrombin generation in-vivo and thus be utilized as an anticoagulant (U.S. Pat. No. 5,120,537, and Benedict, C. R. et al. supra).
Factor Xa can be reversibly inactivated using various derivatives of 4-amidinophenyl benzoate (or p-amidinophenyl ester HCl) acylating compounds which impart reversibility at varying rates. This reversibly inactivated Factor Xa can be used to promote thrombin formation in vivo and thus can be utilized in procoagulant indications (U.S. Pat. No. 4,285,932; U.S. Ser. No. 08/268,003, filed Jun. 26, 1994; and Sinha et al., supra).